Biomedical subjects

Thomas T Chen

Publications and source records attributed to Thomas T Chen.

At least 19 recordsLinked to original sources

PubMed2006

Biological activity of rainbow trout Ea4-peptide of the pro-insulin-like growth factor (pro-IGF)-I on promoting attachment of breast cancer cells (MDA-MB-231) via alpha2- and beta1-integrin.

E-peptide of pro-IGF-I was considered as biologically inactive. We have demonstrated that rainbow trout (rt) Ea4-peptide exerted biological activities in several established tumor cell lines [Chen et al., 2002; Kuo and Chen, 2002]. Here we report the activity of rtEa4-peptide in promoting attachment of human breast cancer cells (MDA-MB-231). While rtEa2-, rtEa3-, and rtEa4-peptides enhanced the attachment of MDA-MB-231 cells in a dose dependent manner, rtEa4-peptide possessed the highest activity. Antibodies specific to alpha2 and beta1 integrins significantly inhibited the attachment of cells to rtEa4-peptide coated-plates by 40%. In addition, rtEa4-peptide induced the expression of fibronectin 1 and laminin receptor genes in MDA-MB-231 cells. Blocking new protein synthesis by cycloheximide significantly reduced the attachment of MDA-MB-231 cells to rtEa4-peptide coated wells by 50%. These results suggest that rtEa4-peptide may promote cell attachment by interacting with alpha2/beta1 integrin receptors at the cell surface and by inducing the expression of fibronectin 1 and laminin receptor genes. Expression of fibronectin 1 gene induced by rtEa4-peptide in MDA-MB-231 cells was abolished by inhibitors of PI3K, PKC, Mek1/2, JNK1/2, and p38 MAPK signaling transduction molecules. These results suggested that induction of fibronectin 1 gene expression in MDA-MB-231 cells by rtEa4-peptide may be mediated via PI3K, PKC, Mek1/2, JNK1/2, and p38 MAPK signal transduction molecules.

Animals↗

PubMed2006

Inhibition of human breast cancer cell (MBA-MD-231) invasion by the Ea4-peptide of rainbow trout pro-IGF-I.

It was shown previously that Ea4-peptide of trout pro-IGF-I exerted mitogenic activity in non-transformed cells and inhibited colony formation in a soft agar medium of established human cancer cells. Here we report that the same peptide inhibits the invasion of human breast cancer cells (MDA-MB-231) through a matrigel membrane in a dose-dependent manner. The expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI1) genes in MDA-MB-231 cells were downregulated by treatment with rtEa4-peptide. The inhibition of expression of these genes in response to rtEa4-peptide treatment was reduced to the control level when inhibitors for c-Jun N-terminal kinase 1/2 (JNK1/2), mitogen activated protein kinase kinase 1/2 (Mek1/2), p38 mitogen activated protein kinase (p38 MAPK), phosphatidylinositol 3-kinase (PI3K), and phosphokinase C (PKC) were used. These results suggest that inhibition of invasion of MDA-MB-231 cells by rtEa4-peptide may be mediated via the suppression of uPA, tPA, and PAI1 gene activities through signal transduction pathways.

Animals↗

PubMed2006

Molecular cloning of myostatin gene and characterization of tissue-specific and developmental stage-specific expression of the gene in orange spotted grouper, Epinephelus coioides.

In this article we report the molecular cloning and characterization of a nonmammalian myostatin (growth and differentiation factor-8, MSTN) homolog from the orange spotted grouper (Epinephelus coioides) by polymerase chain reaction (PCR) cloning. The grouper MSTN gene consists of two introns [Intron I (363 bp) and Intron II (811 bp)] flanked by three exons [Exon I (379 bp), Exon II (371 bp) and Exon III (381 bp)]. A full-length cDNA clone (2608 bp) of the MSTN gene (GenBank DQ493889, nucleotide sequence in the coding region identical to GeneBank AY856860) was also isolated. This cDNA encodes a polypeptide of 376 amino acid residues that showed 25% to 96% homology with MSTNs of molluscan, teleostean, avian, and mammalian species. Phylogenetic analysis of the grouper MSTN polypeptide confirmed the evolutionary relationships of this MSTN with other known MSTNs. Results of reverse transcription (RT)-PCR analysis of the total RNA extracted from different tissues revealed that MSTN gene is expressed not only in the skeletal muscle, but also in other tissues. MSTN mRNA was also detected in different embryonic developmental and larval stages. Because the tissue-specific expression of MSTN gene in grouper is different from that in mammals, it might suggest that MSTN gene may possess additional functions other than regulating muscle growth in fish.

Amino Acid Sequence↗

PubMed2006

Co-induction of hepatic IGF-I and progranulin mRNA by growth hormone in tilapia, Oreochromis mossambiccus.

Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identification and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR amplification, using gene specific oligonucleotides as amplification primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated fish also were substantially induced over controls at 12h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.

Amino Acid Sequence↗

PubMed2006

Microinjecting recombinant rainbow trout Ea4-peptide of pro-IGF-I into zebrafish embryos causes abnormal development in heart, red blood cells, and vasculature.

E-peptides and mature insulin-like growth factors (IGFs) are produced from pre-pro-IGFs during post-translational processing and co-secreted into the circulation. Previously, we reported that introduction of a transgene encoding the secreted form of rainbow trout (rt) Ea4-peptide or human (h) Eb-peptide into newly fertilized eggs of medaka (Oryzias latipes) and zebrafish (Danio rerio) resulted in developmental defects in heart, red blood cells and vasculature. In addition to vasculature and red blood cell developmental defects, multiple phenocopies of heart developmental defects categorized by developmental arrest at cardiomyocyte, heart tube and heart looping stages were also observed. These results raise a question of whether rtEa4- or hEb-peptide exerts pleiotropic inhibitory effects on heart, vasculature and red blood cell development in fish embryos. To answer this question, various amounts of recombinant rtEa4-peptide were microinjected into zebrafish eggs at 1.5, 2.5 and 5.5 h post-fertilization (hpf). Although a dose-dependent developmental defect in heart, vasculature and red blood cells was observed in embryos microinjected with rtEa4-peptide at 1.5 and 2.5 hpf, the heart development in all of the microinjected embryos was arrested at the cardiomyocyte stage. Furthermore, the mRNA levels of Nkx2.5, GATA5, VEGF, GATA1 and GATA2 genes in defective embryos were significantly reduced by rtEa4-peptide. These results confirm our previous findings that rtEa4- or hEb-peptide exhibits pleiotropic effects in inhibiting heart, vasculature and red blood cell development in zebrafish embryos.

Animals↗

PubMed2006

Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages.

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage.

Amino Acid Sequence↗

PubMed2006

Trout Ea4- or human Eb-peptide of pro-IGF-I disrupts heart, red blood cell, and vasculature development in zebrafish embryos.

E-peptide of the pro-insulin-like growth factor (pro-IGF)-I is produced by proteolytic cleavage of the pro-hormone in post-translational processing. Introduction of a transgene encoding a secreted form of rtEa4- or hEb-peptide into newly fertilized zebrafish (Danio rerio) eggs by electroporation or microinjection resulted in embryos with abnormal cardiovascular features and reduced red blood cells and vasculature. Two different phenocopies of heart developmental defects were observed: (i) Group I embryos exhibited heart development arrested at the heart muscle stage and (ii) group II embryos exhibited heart development arrested at the heart tube stage. Both groups of embryos also exhibited reduction of red blood cells and vasculature. The mRNA levels of genes essential for heart development (GATA 5 and NKX2.5), hematopoiesis (GATA 1 and GATA 2), and vasculogenesis (VEGF) in normal and defective embryos were determined by quantitative real-time RT-PCR at 36 hr post-fertilization (hpf). Significant reduction of GATA 5, NKX2.5, GATA 1, GATA 2, and VEGF mRNA levels was observed in both groups of defective embryos. These results suggest that overexpression of rtEa4 or hEb transgene in zebrafish embryos disrupts heart development, hematopoiesis, and vasculogenesis by reducing the levels of GATA 5, NKX2.5, GATA 1, GATA 2, and VEGF mRNA.

Animals↗

PubMed2005

Effects of linear cationic alpha-helical antimicrobial peptides on immune-relevant genes in trout macrophages.

There are increasing evidence of the potential role of antimicrobial peptides in the regulation of immune responses in mammalian species. However, the effects of these peptides in fish have yet to be investigated. In this study, we examined the transcriptional expression profile of representative immune-relevant genes in a trout macrophage cell line, RTS11, in response to three linear cationic alpha-helical antimicrobial peptides (insect cecropin B, fish pleurocidin and a cecropin analogue CF17). The expression levels of two pro-inflammatory genes, interleukin-1 beta (IL-1 beta) and cyclo-oxygenase-2 (COX-2), increased in the peptide-treated RTS11 cells. The peptides did not appear to affect the expression levels of representative genes associated with antigen presentation, interferon response or JAK/STAT signal transduction. Furthermore, the induction of IL-1 beta and COX-2 in RTS11 by lipopolysaccharide was not adversely affected by these three antimicrobial peptides. Overall, the data indicate a pro-inflammatory effect of the three cationic antimicrobial peptides in the inflammatory response of salmonid species, suggesting a potential application of these peptides as immune adjuvant for fish vaccination.

Aeromonas↗

PubMed2005

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis.

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.

Animals↗

PubMed2005

Effects of endocrine disrupters on the expression of growth hormone and prolactin mRNA in the rainbow trout pituitary.

It is now widely accepted that chemical pollutants in the environment can interfere with the endocrine system of animals, thus affecting development and reproduction. Some of these endocrine disrupters (EDs) can have estrogenic or antiestrogenic effects. Most studies to date have focused on the effects of EDs on the reproductive system and sex hormones and only limited information exists on how EDs may affect pituitary gland function. A rainbow trout (Oncorhynchus mykiss) pituitary gland culture system was used for studying the effects of EDs on growth hormone (GH) and prolactin (PRL) mRNA expression. We determined that the pituitary glands actively synthesized and secreted GH and PRL over the experimental time-course. In addition, we found that treatment with 17beta-estradiol (positive control) increased levels of GH and PRL mRNA, in a concentration-dependent manner. Treatment of pituitary glands with 500 and 1000 nM of a xenoestrogen, o,p'-DDT (o,p'-dichlorodiphenyltrichloroethane), resulted in a significant induction of GH and PRL mRNA, with a 20-fold increase for PRL and 3-fold increase for GH following treatment with 1000 nM o,p'-DDT. Co-incubation of pituitary glands with ICI 182 780 (a selective estrogen receptor antagonist) and o,p'-DDT resulted in inhibition of PRL mRNA levels; however, the stimulatory effect of DDT on GH mRNA was not seen in this experiment, nor was the inhibitory effect of ICI 182 780 observed with GH mRNA. To the contrary, ICI 182 780 (2.5 nM) had a stimulatory effect on GH mRNA levels. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which is known to exert antiestrogenic effects, had an estrogenic-like effect that resulted in a concentration-dependent increase in the levels of GH and PRL mRNA. Co-incubation of pituitaries with TCDD and alpha-napthoflavone (ANF), which is an inhibitor of the aryl hydrocarbon receptor (AhR), caused an inhibition of TCDD-induced PRL mRNA at the higher and lower concentrations, but these effects were less consistent on GH mRNA levels. However, the responses of PRL and GH mRNA to co-incubation with TCDD and ANF, at the various concentrations, were bi-phasic wherein stimulation was seen at the low concentrations and inhibition at the high concentrations. Combined, these results suggest that o,p'-DDT and TCDD are xenoestrogens and that their effects on the expression of GH and PRL genes in the rainbow trout pituitary are modulated, in part, through the ER and AhR, respectively.

Animals↗

PubMed2005

Molecular cloning and characterization of cDNA of penaeidin-like antimicrobial peptide from tiger shrimp (Penaeus monodon).

We report the molecular cloning and characterization of a penaeidin-like antimicrobial peptide (AMP) complementary DNA from the hemocytes of tiger shrimp (Penaeus monodoni). A tiger shrimp AMP cDNA containing the entire coding region of the peptide, determined by rapid amplification of cDNA 5' and 3' ends and polymerase chain reaction of the messenger RNA isolated from hemocytes of the shrimp, is 683 bp in length with an open reading frame of 222 bp. The deduced amino acid sequence of this antimicrobial peptide consists of 55 amino acid residues of the mature peptide and a signal peptide of 19 amino acid residues. The mature peptide contains a proline-rich domain at the N terminus and 6 cysteine residues at the C terminus, and it shares less than 50% amino acid sequence identity with the mature penaeidins of Litopenaeus vannamei. To demonstrate the bactericidal activity of this peptide, a synthetic peptide was prepared according to the amino acid sequence deduced from the cDNA of tiger shrimp penaeidin-like AMP. By the minimal inhibitory concentration MIC assay, the synthetic peptide was shown to exert bactericidal activity against Escherichia coli, Vibrio harveyi, Vibrio alginolyticu, and Aerococcus viridans. It also inhibited the growth of 2 filamentous fungi, Fusarium pisi and Fusarium oxysporum. Tiger shrimp penaeidin-like AMP mRNA was detected in hemocytes, gills, intestines, eyestalks, hepatopancreas, and muscles of the tiger shrimp by reverse transcriptase-polymerase chain reaction assay. Although the highest level was detected in hemocytes, challenging tiger shrimp with V. harveyi did not result in a significant increase of the mRNA level in hemocytes.

Amino Acid Sequence↗

PubMed2004

Expression and activation of signal regulatory protein alpha on astrocytomas.

High-grade astrocytomas and glioblastomas are usually unresectable because they extensively invade surrounding brain tissue. Here, we report the expression and function of a receptor on many astrocytomas that may alter both the proliferative and invasive potential of these tumors. Signal regulatory protein (SIRP) alpha1 is an immunoglobulin superfamily transmembrane glycoprotein that is normally expressed in subsets of myeloid and neuronal cells. Transfection of many cell types with SIRPalpha1, including glioblastomas, has been shown to inhibit their proliferation in response to a range of growth factors. Furthermore, the expression of a murine SIRPalpha1 mutant has been shown to enhance cell adhesion and initial cell spreading but to inhibit cell extension and movement. The extracellular portion of SIRPalpha1 binds CD47 (integrin-associated protein), although this interaction is not required for integrin-mediated activation of SIRPalpha1. On phosphorylation, SIRPalpha1 recruits the tyrosine phosphatases SHP-1 and SHP-2, which are important in its functions. Although SHP-1 is uniquely expressed on hematopoietic cells, SHP-2 is ubiquitously expressed, so that SIRPalpha1 has the potential to function in many cell types, including astrocytomas. Because SIRPalpha1 regulates cell functions that may contribute to the malignancy of these tumors, we examined the expression of SIRPs in astrocytoma cell lines by flow cytometry using a monoclonal antibody against all SIRPs. Screening of nine cell lines revealed clear cell surface expression of SIRPs on five cell lines, whereas Northern blotting for SIRPalpha transcripts showed mRNA present in eight of nine cell lines. All nine cell lines expressed the ligand for SIRPalpha1, CD47. To further examine the expression and function of SIRPs, we studied the SF126 and U373MG astrocytoma cell lines, both of which express SIRPs, in greater detail. SIRP transcripts in these cells are identical in sequence to SIRPalpha1. The expressed deglycosylated protein is the same size as SIRPalpha1, but in the astrocytoma cells, it is underglycosylated compared with SIRPalpha1 produced in transfected Chinese hamster ovary cells. It is nonetheless still capable of binding soluble CD47. Moreover, SIRPalpha1 in each of the two cell lines recruited SHP-2 on phosphorylation, and SIRPalpha1 phosphorylation in cultured cells is CD47 dependent. Finally, examination of frozen sections from 10 primary brain tumor biopsies by immunohistochemistry revealed expression of SIRPs on seven of the specimens, some of which expressed high levels of SIRPs. Most of the tumors also expressed CD47. This is the first demonstration that astrocytomas can express SIRPalpha. Given the known role of SIRPalpha in regulating cell adhesion and responses to mitogenic growth factors, the expression of SIRPalpha1 on astrocytomas may be of considerable importance in brain tumor biology, and it offers the potential of a new avenue for therapeutic intervention.

Antigens, Differentiation↗

PubMed2004

Up-regulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock-down of myostatin-1.

Myostatin, a secreted growth and differentiation factor (GDF-8) belongs to transforming growth factor (TGF-beta) superfamily that plays as a negative regulator of skeletal muscle development and growth. Recently, myostatin has been isolated from fish; however, its role in muscle development and growth remains unknown. Here, we present the expression of myostatin during development and the effects of its knock-down on various genes such as muscle regulatory transcription factors (MRFs), muscle-specific proteins (MSP), and insulin-like growth factors (IGFs). The myostatin expression was found to be maternal as it starts in one-cell stage onward. The reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and Southern and Northern blots demonstrated that the myostatin expression is not only restricted to skeletal muscle, but it expressed all the tested tissues. Expression of myostatin was effected by using antisense morpholinos resulted in significant phenotypic difference in stages 18 and 20 hours postfertilization (hpf). To confirm the specificity of myostatin morpholino, furthermore, a rescue experiment was conducted. The length as well as width of somites was increased with almost no gap in between the somites. In addition, it deserves to mention that this is a first animal model that shows changes in the size of the somites. Moreover, analyses of MRFs, MSP, and IGFs in the knock-down embryos by RT-PCR revealed the up-regulation of MyoD, Myogenin, and Mck transcription, whereas IGF-2 transcription showed mild response with no effect on IGF-1, Desmin, and Myf5. In situ hybridization showed that there was an increase in the number of somites from 3 to 4 at 13 and 22 hpf. Taken together, these data suggest that myostatin plays a major role during myogenesis, apart from inhibition of proliferation as well as differentiation.

Animals↗

PubMed2003

Molecular cloning and tissue-specific, developmental-stage-specific, and hormonal regulation of IGFBP3 gene in zebrafish.

The biological functions of insulin-like growth factor (IGF) I and II are modulated by a family of IGF-binding proteins (IGFBPs) in complex IGF-dependent and IGF-independent pathways. For further understanding of the actions of IGFs, some of these binding proteins have been cloned and characterized. We report the molecular cloning of IGFBP-3 cDNA for zebrafish. The tissue-specific and developmental stage-specific expression of IGFBP-3 and the hormonal regulation of its expression have also been determined by comparative reverse transcription polymerase chain reaction. Zebrafish IGFBP-3 cDNA contains an open reading frame of 879 bp, encoding a polypeptide of 293 amino acid residues. Results of this analysis revealed high levels of IGFBP-3 messenger RNA in ovary and fin tissue. Expression of IGFBP-3 mRNA was throughout the entire embryonic development, with the highest level of expression observed at 36 hours after the onset of development. Elevated levels of expression of IGFBP-3 were observed 24 hours after injection with IGF-I and 48 hours with IGF-II or insulin. These results suggest that the expression of IGFBP3 gene might be modulated by IGF-I, IGF-II, and insulin.

Amino Acid Sequence↗

PubMed2003

Pattern recognition by TREM-2: binding of anionic ligands.

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.

Animals↗

PubMed2003

Specific cell surface binding sites shared by human Pro-IGF-I Eb-peptides and rainbow trout Pro-IGF-I Ea-4-peptide.

Human pro-IGF-I Eb-peptide (hEb) and rainbow trout pro-IGF-I Ea-4-peptide (rtEa-4) have recently been shown to share unique biological activities [Gen. Comp. Endocrinol. 126 (2002) 342; Cell. Exp. Cell. Res. 280 (2002) 75]. To further understand the action mechanism of these proteins, we studied the binding properties of hEb-peptide and rtEa-4-peptide to intact human neuroblastoma cells (SK-N-F1) and membrane preparations. Human Eb-peptide and rtEa-4-peptide bind to a high-affinity binding site with an apparent dissociation constant of 3.2+/-1.9 x 10(-11) and 2.9+/-1.8 x 10(-11)M, respectively. Homologous displacement assay demonstrated the presence of a second binding site with an IC(50) of 4.8+/-2.6 x 10(-6)M for hEb-peptide and 2.1+/-0.6 x 10(-6)M for rtEa-4-peptide, respectively. Competition assays showed that hEb-peptide and rtEa-4-peptide shared common binding sites, distinct from those for IGF-I and insulin. In addition, chemical cross-linking studies revealed two specific binding complexes. Our findings support the notion that the initial step of pro-IGF-I E-peptide action is mediated through the interaction with conserved and specific putative membrane receptors on neuroblastoma cells.

Animals↗

PubMed2003

Identification of a new growth hormone family protein, somatolactin-like protein, in the rainbow trout (Oncorhyncus mykiss) pituitary gland.

A cDNA encoding a new GH family protein expressed in the rainbow trout (Oncorhyncus mykiss) pituitary gland was identified. Because the deduced amino acid sequence of this cDNA shares 56% homology with rainbow trout (rt) somatolactin (rtSL), we named it as rtSL-like protein (rtSLP). Comparison of the amino acid sequences of rtGH, rainbow trout prolactin (rtPRL), and rtSLP revealed that rtSLP shares 26% and 21% identity with that of rtGH and rtPRL, respectively. Trout SLP contains not only the known GH family protein conserved domains but also all of the four cysteine residues that are responsible for the formation of two disulfide linkages in GH, PRL, and SL. Immunoblot analysis revealed that rtSLP is secreted from the pituitary gland once it is synthesized. Unlike rtSL, rtSLP mRNA was readily detectable in pituitary glands of 2-yr fish (30-34 cm in length) but not in embryos or 1-yr fish (17-25 cm in length). Furthermore, the level of rtSLP mRNA in male fish is 2-fold higher than that in female fish. These results suggest that rtSLP might play a role in regulating the reproductive maturation in rainbow trout.

Amino Acid Sequence↗

PubMed2002

Novel activities of pro-IGF-I E peptides: regulation of morphological differentiation and anchorage-independent growth in human neuroblastoma cells.

Insulin-like growth factor-I (IGF-I) is translated as a pre-pro-peptide that is posttranslationally processed to its mature form by proteolytic removal of the signal peptide and the E-domain peptide. Contrary to the mature human (h) IGF-I, the recombinant rtEa4 -peptide significantly reduced the anchorage-independent cell growth in human neuroblastoma cells (SK-N-F1), shown by colony formation assay in vitro. Significant inhibition of colony formation is also observed in SK-N-F1 cells stably transfected with a bicistronic expression construct encoding a secretory form of the rtEa4 peptide. Furthermore, treatment with the recombinant rtEa4 peptide, but not the mature hIGF-I, resulted in morphological differentiation of SK-N-F1 cells characterized by long neurite outgrowth. Similar morphological differentiation is also observed in SK-N-F1 cell clones stably transfected with the rtEa4 peptide expression construct. A spectrum of biological activities similar to those of rtEa4 peptide is also observed in the synthetic hEb peptide, but not-the hEa peptide. Results of further characterization reveal that neurites induced by rtEa4 or hEb peptide contain neuronal-specific MAP-2, Tau, and neurofilament (NF-160), accompanied by an increased expression of the neuronal marker gene neuropeptide tyrosine (NPY). Furthermore, effects of signal transduction inhibitors are indicative of the involvement of MAP-kinase PI-3-kinase cascades. The activation of ERK-1/-2 is markedly increased in response to rtEa-4 or hEb peptide stimulation, further indicating the involvement of MAPK signaling cascade. These unique biological activities exhibited by the rtEa4 or hEb peptide suggest that E peptide of the pro-IGF-I may play distinct roles in regulating cell growth and differentiation in neuroblastoma cells.

Biomarkers↗